Serine proteinase inhibitors

ABSTRACT

The invention relates to serine protease inhibitors having the amino acid sequence in accordance with Seq. ID No. 1.

The present invention relates to serine protease inhibitors, cDNA codingfor serine protease inhibitors, medicaments containing such inhibitorsor their coding nucleic acid, use of the compounds according to theinvention for the preparation of medicaments for the treatment ofvarious indications, antibodies or antibody fragments against epitopesof the compounds according to the invention, poly- or oligonucleotideswhich will hybridize to genes of the compounds according to theinvention, a diagnostic agent for detecting the compounds according tothe invention, and medicaments containing antibodies or poly- oroligonucleotides according to the invention.

Proteolytic processes play an important physiological role in allorganisms; a distinction has to be made between non-specific andspecific proteolytic reactions. The former include, for example, thedigestion of food in the digestive tract by endopeptidases, and theintracellular degradation of used endogenous substances and phagocytosedmaterials by lysosomal proteases. Specific proteolyses mostly serve forthe conversion of a proenzyme to its active form, as in the conversionof trypsinogen to trypsin, and of chymotrypsinogen to chymotrypsin, andin the callicrein-kinin cascades and the blood clotting cascade.Depending on the structure of the reactive site of the proteinasesinvolved, they are classified into the classes of serine proteases(e.g., chymotrypsin, trypsin, elastase and cathepsin G), aspartateproteases (e.g., cathepsin D, cathepsin E and pepsin), cysteineproteases (e.g., cathepsin B, cathepsin H and cathepsin L), and themetallo-proteases (e.g., collagenase and thermolysin).

In order to be able to correct the proteolytic processes which oftenproceed in a cascade, the organisms is provided with a number of otherproteins, the protease inhibitors (for a survey, see Laskowski and Kato,1980, and Bode and Huber, 1992). Thus, the liver-synthesized humanplasma protease inhibitors α₁-antichymotrypsin and α₁-proteinaseinhibitors protect the lung tissue from non-specific attack by theproteinases cathepsin G and elastase from polymorphonuclear lymphocytes.When the balance between proteases and their specific inhibitors isdisturbed, pathological effects may arise. For example, an excess ratioof elastase to α₁-proteinase inhibitor increases the risk of formationof a lung emphysema by a factor of about 20 to 30 in patients with agenetically caused deficiency in this factor as compared to the normalpopulation (Carrel and Owen, 1980). With smokers, the formation of anemphysema is promoted by oxidation of the amino acid methionine which ispresent in the reactive site of the α₁-proteinase inhibitor by oxidantscontained in cigarette smoke (Miller and Kuschner, 1969; Ohlsson et al.,1980). Also in the case of infection with Gram-negative bacteria, theirendotoxins can cause disintegration of phagocytes and thus the secretionof lysosomal proteases, which may cause an uncontrolled damage totissues and inflammations due to the increased consumption of proteaseinhibitors. For this reason, certain protease inhibitors have a hightherapeutic potential (see, e.g., Fritz, 1980).

International Application PCT/EP 98/08424 relates to serine proteaseinhibitors, wherein said serine protease inhibitors have a domain withfour cysteines, and a sequence of from 0 to 20 amino acids is presentbetween the first and second cysteines, or said serine proteaseinhibitors have a domain of six cysteines, and a sequence of from 7 to20 amino acids is present between the first and second cysteines.

BRIEF SUMMARY OF THE INVENTION

It has been the object of the present invention to provide furtherserine protease inhibitors.

This object is achieved by a serine protease inhibitor having the aminoacid sequence according to SEQ ID NO: 1.

The present invention also relates to fragments of the serine proteaseinhibitor according to the invention having the amino acid sequenceR₁—X—R₂, wherein R₁ is NH₂, an amino acid or a peptide with up to 100amino acids, and R₂ is COOH, CONH₂, an amino acid or a peptide with upto 100 amino acids, and X is selected from SEQ ID NOS: 2 to 6.

It is preferred that the serine protease inhibitor contains one or moredisulfide bridges. It is particularly preferred for it to contain adisulfide bridge between the first and fourth cysteines and/or betweenthe second and third cysteines, or to contain a disulfide bridge betweenthe first and fifth cysteines and/or between the second and fourthcysteines and/or between the third and sixth cysteines.

In addition to the amino acid sequence of the preferred compoundsaccording to the invention, further information about the cDNA codingfor the compounds according to the invention can also be seen from FIG.1. In particular, the corresponding motifs and primer-hybridizing sitesare indicated.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates, in addition to the amino acid sequence of thepreferred compounds according to the invention, further informationabout the cDNA coding for the compounds according to the invention. Inparticular, the corresponding motifs and primer- hybridizing sites areindicated.

According to the invention, nucleic acids coding for the compoundsaccording to the invention, especially a DNA having the nucleic acidsequence according to SEQ ID NOS: 7 to 12, are also claimed.

The compounds according to the invention are useful as medicaments. Inthis case, they are administered together with pharmaceuticallyacceptable vehicles.

The medicaments according to the invention containing the proteaseinhibitors according to the invention are preferably administered inamounts of from 1 to 100 mg/kg of the patient's body weight. As thedosage form, all galenic formulations for peptide active substances maybe used. The medicaments containing nucleic acids according to theinvention are preferably administered in amounts of from 0.1 to 100mg/kg of body weight of a corresponding patient. In this case, thegalenic dosage forms which may be used are those which are suitable forthe administration of nucleic acids without rendering the nucleic acidsineffective by metabolic influences before they have reached their siteof action. For example, liposomes in which the nucleic acids arecontained can be employed as a galenic dosage form.

The compounds according to the invention can be used, in particular, forthe treatment of acute or chronic cervix inflammations, inflammations ofBartholin's gland or other vaginal regions, tonsillitis, pharyngitis andlaryngitis, acute or chronic inflammatory processes accompanied byexcessive formation of mucus and the resulting acute emergencysituations, postoperative bleedings due to hyperfibrinolysis, and forthe prophylaxis of lung emphysema formation in deficiencies ofα₁-proteinase inhibitor.

Further, they may be employed for the therapy of asthma, AIDS, tumordiseases and leukemia.

The compounds according to the invention can be administered indeficiencies of serine protease inhibitors to correct endogenousdefects. The nucleic acids may also be used in gene therapy, eitherdirectly or coupled to suitable vehicles. Suitable vectors include, inparticular, attenuated adenoviruses into which the corresponding geneshave been incorporated.

The polypeptides according to the invention can serve for thepreparation of antibodies or antibody fragments. These are simplyprepared by the immunization of appropriate mammals. By per se knownoperations, the antibodies may also be humanized so that such antibodiescan also be employed for therapeutic use. Antibodies or antibodyfragments can then by employed for the regulation of diseases in whichthe protease inhibitors are expressed in a pathological way. Also,antisense nucleic acids complementary to the nucleic acids according tothe invention may be employed in therapeutical use in overexpressions ofthe protease inhibitor genes.

The compounds according to the invention can be easily prepared by perse known methods of peptide or nucleotide synthesis. Preparation of thecompounds by genetic engineering is also possible.

Those skilled in the art will recognize that fragments of thepolypeptides according to the invention may also be used provided thatthey retain the inhibitory properties of the serine protease inhibitors.Those skilled in the art know how to find such fragments. Thus, this maybe accomplished, for example, by a selected enzymatic cleavage of thecompounds according to the invention. Side-chain modified amino acidsmay also be employed. N- or C-terminally modified polypeptides may alsobe used. In particular, phosphorylated, glycosylated, methylated,acetylated or similarly modified polypeptides can be employed providedthat they do not substantially affect the activity of the serineprotease inhibitors.

Derivatives of the nucleic acids according to the invention which havemodified triplet structures in accordance with codon usage may also beused. In addition, nucleic acids according to the invention also includethose which are more stable towards degradation by nucleases as comparedwith the native compounds, for example, the corresponding SODNderivatives usually employed in antisense technology to give theantisense structures a more stable design towards enzymatic attack.

Structures homologous to the polypeptides may also be used. Inparticular, these include polypeptide structures in which amino acidshave been exchanged. Thus, for example, conservative amino acidsubstitutions in highly conserved regions can be considered as follows:any isoleucine, valine and leucine amino acid can be exchanged for anyother of these amino acids, aspartate can be exchanged for glutamate andvice versa, glutamine for asparagine and vice versa, serine forthreonine and vice versa. Conservative amino acid substitutions in lesshighly conserved regions can be as follows: Any of the amino acidsisoleucine, valine and leucine for any other of these amino acids,aspartate for glutamate and, vice versa, glutamine for asparagine andvice versa, serine for threonine and vice versa, glycine for alanine andvice versa, alanine for valine and vice versa, any of the amino acidsleucine, isoleucine or valine for methionine, lysine for arginine andvice versa, either of the amino acids arginine or lysine for either ofthe amino acids aspartate or glutamate, either of the amino acidsarginine or lysine for histidine, glutamine for glutamate and viceversa, and asparagine for aspartate and vice versa.

1. A serine protease inhibitor having the amino acid sequence accordingto SEQ ID NO:
 1. 2. A medicament containing at least one serine proteaseinhibitor according to claim 1, optionally together with apharmaceutical vehicle.
 3. The medicament according to claim 2,containing from 0.01 to 1000 mg of the serine protease inhibitor per kgof body weight of a patient in need thereof.
 4. A method of using theserine protease inhibitor according to claim 1 comprising administeringthe serine protease inhibitor to a patient in need thereof as amedicament for the treatment of acute or chronic cervix inflammations,inflammations of Bartholin's glands or other vaginal regions,tonsillitis, pharyngitis or laryngitis, or acute or chronic inflammatoryprocesses accompanied by excessive formation of mucus the resultingacute emergency situations, postoperative bleedings due tohyperfibrinolysis or for the prophylaxis of lung emphysema formationbecause of deficiencies of α₁-proteinase inhibitor.